Methods and compositions for modulating angiogenesis and vasculogenesis

ABSTRACT

Disclosed herein are methods and compositions for stimulating angiogenesis, using cells descended from marrow adherent stromal cells that have been transfected with sequences encoding a Notch intracellular domain. Applications of these methods and compositions include treatment of ischemic disorders such as stroke.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/063,290 filed Mar. 7, 2016 which is a continuation of U.S. patentapplication Ser. No. 13/750,772 filed Jan. 25, 2013 which claims thebenefit of U.S. Provisional Patent Application No. 61/591,486 filed onJan. 27, 2012, U.S. Provisional Patent Application No. 61/637,740 filedon Apr. 24, 2012, and U.S. Provisional Patent Application No. 61/709,619filed on Oct. 4, 2012. The specifications and drawings of all of theaforementioned applications are incorporated herein by reference in thenentireties for all purposes.

STATEMENT REGARDING FEDERAL SUPPORT

Not applicable.

FIELD

The present disclosure is in the fields of angiogenesis andvasculogenesis; e.g., for the treatment of ischemic events such asstroke. It is also in the field of stem cells and cells derived fromstem cells by genetic manipulation.

BACKGROUND

In stable stroke, reinstating vascular flow is imperative for restoringnutrient supply in the brain. To repair vascular damage after prolongedischemia at least two sequential steps are needed. The first step isangiogenic sprouting of endothelial cells (ECs); this process entailsthe initial proliferation of endothelial cells and remodeling of thesurrounding extracellular matrix. VEGF-mediated prolifdation of ECs andmatrix metalloproteinases are among the major components of angiogenicsprouting. The second step is vessel stabilization; a process thatrelies on recruitment of vascular smooth muscle cells to encase theyoung vessels. Monocytes and pericytes are also involved in vesselstabilization, producing the appropriate arteriogenic factors andextracellular matrix proteins. In the absence of vessel stabilization bysmooth muscle cells and pericytes, regression of nascent vasculature canoccur.

Marrow stromal cells (MSCs, also known as mesenchymal stem cells)) havebeen shown to promote revascularization after cerebral artery occlusionand traumatic brain in jury. Omori et al. (2008) Brain Res. 1236:30-38;Onda et al. (2008) J. Cereb. Blood Flow Metab. 28:329-340; Pavlichenkoet al. (2008) Brain Res. 1233:203-213; Xiong et al.(2009) Brain Res.1263:183-191. SB623 cells are a derivative of marrow stromal cells,obtained by transfecting marrow stromal cells with a vector containingsequences encoding a Notch intracellular domain (NICD). See, forexample, U.S. Pat. No. 7,682,825 and Dezawa et al. (2004) J. Clin.Investig. 13:1701-1710. SB623 cells elicit functional improvement inexperimental stroke models. See, for example, U.S. Pat. No. 8,092,792and Yasuhara et al. (2009) Stem Cells and Development 18:1501-1514.Although the secretome of SB623 cells is comparable to that of theparental MSCs; different levels of specific trophic factors have beenobserved to be secreted by MSCs, as compared to SB623 cells. See, forexample, Tate et al. (2010) Cell Transplantation 19:973-984; U.S. PatentApplication Publication No. 2010/0266554. Moreover, many of the factorswhose expression levels differ between MSCs and SB623 cells have beenreported to be involved in vascular regeneration.

Because stroke is a leading cause of adult disability in the UnitedStates, and is the second leading cause of death worldwide, thereremains a need for treatments to restore blood supply to, and promotereperfusion of, regions of stroke-induced ischemic damage in the brain.

SUMMARY

The present inventors have discovered that descendents of mesenchymalstein cells that have been transfected with sequences encoding a Notchintracellular domain (i.e. SB623 cells) have the surprising property ofbeing able to synthesize and secrete factors that promote angiogenesis.Because angiogenesis, i.e., the formation of new blood vessels, is acritical part of the endogenous repair process in brain injury anddisease, this discovery provides new methods of treatment for vasculardisorders such as stroke.

Accordingly, the present disclosure provides, inter alia:

1. A method for augmenting angiogenesis in a subject, the methodcomprising administering to the subject a population of SB623 cells;wherein the SB623 cells, are obtained by (a) providing a culture ofmesenchymal stem cells, (b) contacting the cell culture of step (a) witha polynucleotide comprising sequences encoding a Notch intracellulardomain (NICD) wherein said polynucleotide does not encode a full-lengthNotch protein, (c) selecting cells that comprise the polynucleotide ofstep (b), and (d) further culturing the selected cells of step (c) inthe absence of selection.

2. The method of embodiment 1, wherein the augmentation of angiogenesisoccurs in the central nervous system.

3. The method of embodiment 2, wherein the augmentation of angiogenesisoccurs in the brain.

4. A method for repairing ischemic damage in a subject, the methodcomprising administering to the subject a population of SB623 cells;wherein the SB623 cells are obtained by (a) providing a culture ofmesenchymal stem cells, (b) contacting the cell culture of step (a) witha polynucleotide comprising sequences encoding a Notch intracellulardomain (NICD) wherein said polynucleotide does not encode a full-lengthNotch protein, (c) selecting cells that comprise the polynucleotide ofstep (b), and (d) further culturing the selected cells of step (c) inthe absence of selection.

5. The method of embodiment 4, wherein the ischemic damage occurs in thecentral nervous system.

6. The method of embodiment 5, wherein the ischemic damage occurs in thebrain.

7. The method of embodiment 6, wherein the ischemic damage results fromstroke.

8. A method for enhancing survival of endothelial cells, the methodcomprising contacting the endothelial cells with a population of SB623cells; wherein the SB623 cells are obtained by (a) providing a cultureof mesenchymal stem cells, (b) contacting the cell culture of step (a)with a polynucleotide comprising sequences encoding a Notchintracellular domain (NICD) wherein said polynucleotide does not encodea full-length Notch protein, (c) selecting cells that comprise thepolynucleotide of step (b), and (d) further culturing the selected cellsof step (c) in the absence of selection.

9. The method of embodiment 8, wherein the method prevents the death ofendothelial cells.

10. The method of either of embodiments 8 or 9, wherein the endothelialcells are in a subject.

11. The method of embodiment 10, wherein the endothelial cells are in hecentral nervous system of the subject.

12. The method of embodiment 11, wherein the endothelial cells are inthe brain of the subject.

13. A method for stimulating proliferation of endothelial cells, themethod comprising contacting the endothelial cells with a population ofSB623 cells; wherein the SB623 cells are obtained by (a) providing aculture of mesenchymal stem cells, (b) contacting the cell culture ofstep (a) with a polynucleotide comprising sequences encoding a Notchintracellular domain (NICD) wherein said polynucleotide does not encodea full-length Notch protein, (c) selecting cells that comprise thepolynucleotide of step (b), and (d) further culturing the selected cellsof step (c) in the absence of selection.

14. The method of embodiment 13, wherein the endothelial cells are in asubject.

15. The method of embodiment 14, wherein the endothelial cells are inthe central nervous system of the subject.

16. The method of embodiment 15, wherein the endothelial cells are inthe brain of the subject.

17. A method for enhancing the branching of blood vessels, the methodcomprising contacting the vessels with a population of SB623 cellswherein the SB623 cells are obtained by (a) providing a culture ofmesenchymal stem cells, (b) contacting the cell culture of step (a) witha polynucleotide comprising sequences encoding a Notch intracellulardomain (NICD) wherein said polynucleotide does not encode a full-lengthNotch protein, (c) selecting cells that comprise the polynucleotide ofstep (b), and (d) further culturing the selected cells of step (c) inthe absence of selection.

18. The method of embodiment 17, wherein the blood vessels are in asubject

19. The method of embodiment 18, wherein the blood vessels are in thecentral nervous system of the subject.

20. The method of embodiment 19, wherein the blood vessels are in thebrain of the subject.

21. A method for augmenting angiogenesis in a subject, the methodcomprising administering to the subject (1) a population of SB623 cells;wherein the SB623 cells are obtained by (a) providing a culture ofmesenchymal stem cells, (b) contacting the cell culture of step (a) witha polynucleotide comprising sequences encoding a Notch intracellulardomain (NICD) wherein said polynucleotide does not encode a full-lengthNotch protein, (c) selecting cells that comprise the polynucleotide ofstep (b), and (d) further culturing the selected cells of step (c) inthe absence of selection; and (2) a pro-angiogenic agent.

22. The method of embodiment 21, wherein the augmentation ofangiogenesis occurs in the central nervous system.

23. The method of embodiment 22, wherein the augmentation f angiogenesisoccurs in the brain.

24. The method of embodiment 21, wherein the pro-angiogenic agent is anucleic acid.

25. The method of embodiment 21, wherein the pro-angiogenic agent is apolypeptide.

26. The method of embodiment 25, wherein the polypeptide is atranscription factor that activates expression of a pro-angiogenicprotein.

27. The method of embodiment 26, wherein the pro-angiogenic protein isvascular endothelial growth factor (VEGF).

28. The method of embodiment 27, wherein the transcription factor is anon-naturally-occurring zinc finger protein that activates transcriptionof the VEGF gene.

29. A method for repairing ischemic damage in a subject, the methodcomprising administering to the subject (1) a population of SB623 cells;wherein the SB623 cells are obtained by (a) providing a culture ofmesenchymal stem cells, (b) contacting the cell culture of step (a) witha polynucleotide comprising sequences encoding a Notch intracellulardomain (NICD) wherein said polynucleotide does not encode a full-lengthNotch protein, (c) selecting cells that comprise the polynucleotide ofstep (b), and (d) further culturing the selected cells of step (c) inthe absence of selection; and (2) a pro-angiogenic agent.

30. The method of embodiment 29, wherein the ischemic damage occurs inthe central nervous system.

31. The method of embodiment 30, wherein the ischemic damage occurs inthe brain.

32. The method of embodiment 31, wherein the ischemic damage resultsfrom stroke.

33. The method of embodiment 29, wherein the pro-angiogenic agent is anucleic acid.

34. The method of embodiment 29, wherein the pro-angiogenic agent is apolypeptide.

35. The method of embodiment 34, wherein the polypeptide is atranscription factor that activates expression of a pro-angiogenicprotein.

36. The method of embodiment 35, wherein the pro-angiogenic protein isvascular endothelial growth factor (VEGF).

37. The method of embodiment 36, wherein the transcription factor is anon-naturally-occurring zinc finger protein that activates transcriptionof the VEGF gene.

38. A method for treating stroke in a subject, the method comprisingadministering to the subject (1) a population of SB23 cells; wherein theSB623 cells are obtained by (a) providing a culture of mesenchymal stemcells, (b) contacting the cell culture of step (a) with a polynucleotidecomprising sequences encoding a Notch intracellular domain (NICD)wherein said polynucleotide does not encode a full-length Notch protein,(c) selecting cells that comprise the polynucleotide of step (b), and(d) further culturing the selected cells of step (c) in the absence ofselection; and (2) a pro-angiogenic agent.

39. The method of embodiment 38, wherein the pro-angiogenic agent is anucleic acid.

40. The method of embodiment 38, wherein the pro-angiogenic agent is apolypeptide.

41. The method of embodiment 40, wherein the polypeptide is atranscription factor that activates expression of a pro-angiogenicprotein.

42. The method of embodiment 41, wherein the pro-angiogenic protein isvascular endothelial growth factor (VEGF).

43. The method of embodiment 42, wherein the transcription factor is anon-naturally-occurring zinc finger protein that activates transcriptionof the VEGF gene.

44. The method of any of embodiments 8, 9, or 13, further comprisingadministering a pro-angiogenic agent along with the SB623 cells.

45. The method of embodiment 44, wherein the endothelial cells are in asubject.

46. The method of embodiment 45, wherein the endothelial cells are inthe central nervous system of the subject.

47. The method of embodiment 46, wherein the endothelial cells are inthe brain of the subject.

48. The method of embodiment 44, wherein the pro-angiogenic agent is anucleic acid.

49. The method of embodiment 44, wherein the pro-angiogenic agent is apolypeptide.

50. The method of embodiment 49, wherein the polypeptide is atranscription factor that activates expression of a pro-angiogenicprotein.

51. The method of embodiment 50, wherein the pro-angiogenic protein isvascular endothelial growth factor (VEGF).

52. The method of embodiment 51, wherein the transcription factor is anon-naturally-occurring zinc finger protein that activates transcriptionof the VEGF gene.

53. The method of embodiment 17, further comprising administering apro-angiogenic agent along with the SB623 cells.

54. The method of embodiment 53, wherein the blood vessels are in asubject.

55. The method of embodiment 54, wherein the blood vessels are in thecentral nervous system of the subject.

56. The method of embodiment 55, wherein the blood vessels are in thebrain of the subject.

57. The method of embodiment 53, wherein the pro-angiogenic agent is anucleic acid.

58. The methot f embodiment 53, wherein the pro-angiogenic agent s apolypeptide.

59. The method of embodiment 58, wherein the polypeptide is atranscription factor that activates expression of a pro-angiogenicprotein.

60. The method of embodiment 59, wherein the pro-angiogenic protein isvascular endothelial growth factor (VEGF).

61. The method of embodiment 60, wherein the transcription factor is anon-naturally-occurring zinc finger protein that activates transcriptionof the VEGF gene.

62. A method for providing an angiogenic factor to a subject, whereinthe method comprises administering to the subject a population of SB623cells; wherein the SB623 cells are obtained by (a) providing a cultureof mesenchymal stem cells, (b) contacting the cell culture of step (a)with a polynucleotide comprising sequences encoding a Notchintracellular domain (NICD) wherein said polynucleotide does not encodea full-length Notch protein, (c) selecting cells that comprise thepolynucleotide of step (b), and (d) further culturing the selected cellsof step (c) in the absence of selection.

63. The method of embodiment 62, wherein the subject is suffering froman ischemic disorder.

64. The method of embodiment 63, wherein the subject is suffering from adisease or disorder of the central nervous system.

65. The method of embodiment 62, wherein the trophic factor is selectedfrom the group consisting of one or more of angiogenin, angiopoietin-2,epidermal growth factor, basic fibroblast growth factor, heparin-bindingepithelial growth factor-like growth factor, hepatocyte growth factor,leptin, platelet-derived growth factor-BB, placental growth factor andvascular endothelial growth factor.

66. The method of embodiment 65, wherein the trophic factor is vascularendothelial growth factor.

67. A method for providing vascular endothelial growth factor to asubject, wherein the method comprises administering to the subject apopulation of SB623 cells; wherein the SB623 cells are obtained by (a)providing a culture of mesenchymal stem cells, (b) contacting the cellculture of step (a) with a polynucleotide comprising sequences encodinga Notch intracellular domain (NICD) wherein said polynucleotide does notencode a full-length Notch protein, (c) selecting cells that comprisethe polynucleotide of step (b), and (d) further culturing the selectedcells of step (c) in the absence of selection.

68. The method of embodiment 67, wherein the subject is suffering froman ischemic disorder.

69. The method of embodiment 68, wherein the subject is suffering from adisease or disorder of the central nervous system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows measurements of the fraction of cells permeable topropidium iodide in cultures of HUVECs that have been starved for semmand growth factors. Left-most bar shows results obtained from controlserum/growth factor-starved HUVECs; center bar shows results forserum/growth factor-starved HUVECs cultured for seven days in thepresence of conditioned medium from MSCs, and the right-most bar showsresults for serum/growth factor-starved HUVECs cultured for seven daysin the presence of conditioned medium from SB623 cells. Values shown aremean±SD for three separate donors of MSCs and SB623 cells; * indicatesp<0.05 compared to control group.

FIG. 2 shows measurement of the fraction of cells expressing Bcl-2 incultures of HUNTE.Cs that have been starved for serum and growthfactors. Left-most bar shows results obtained from control serum/growthfactor-starved HUVECs; center bar shows results for serum/growthfactor-starved HUVECs. cultured for seven days in the presence ofconditioned medium from MSCs, and the right-most bar shows results forserum/growth factor-starved HUVECs cultured for seven days in thepresence of conditioned medium from SB623 cells. Results were obtainedby measuring fluorescence of cells stained with a fluorescein-conjugatedanti-Bcl-2 antibody and subtracting fluorescence of cells exposed tofluorescein-conjugated IgG. Values shown are mean±SD for three separatedonors of MSCs and SB623 cells; * indicates p<0.05 compared to controlgroup.

FIG. 3 shows measurement of the fraction of cells expressing Ki67 incultures of HUVECs that have been starved for serum and growth factors.Left-most bar shows results obtained from control serum/growthfactor-starved HUVECs; center bar shows results for serum/growthfactor-starved HUVECs cultured for seven days in the presence ofconditioned medium from MSCs, and the right-most bar shows results forserum/growth factor-starved HUVECs cultured for seven days in thepresence of conditioned medium from SB623 cells. Results were obtainedby measuring fluorescence of cells stained with a fluorescein-conjugatedanti-Ki67 antibody and subtracting fluorescence of cells exposed tofluorescein-conjugated IgG. Values shown are mean±SD for three separatedonors of MSCs and SB623 cells; * indicates p<0.05 compared to controlgroup.

FIG. 4 shows phase-contrast photomicrogaphs of HUVECs following culturefor 16 hours in conditioned media from MSCs or SB623 cells. Theleft-most photograph shows cells cultured in conditioned medium fromMSCs; the center photograph shows cells cultured in conditioned mediumfrom SB623 cells; and the right-most photograph shows cells cultured incommercial culture medium without added conditioned medium.

FIG. 5 shows measurement of the effect of conditioned medium on tubeformation by HUVECs. Left-most bar shows results obtained from controlserum/growth factor-starved HUVECs; center bar shows results forsenimigrowth factor-starved HUVECs cultured for 16 hours in the presenceof conditioned medium from MSCs, and the right-most bar shows resultsfor senimigrowth factor-starved HUVECs cultured for 16 hours in thepresence of conditioned medium from SB623 cells. Values shown aremean+SEM for three separate donors of MSCs and SB623 cells.

FIGS. 6A-6C show photographs of aortic rings after culture for 10 daysin unconditioned medium (A), MSC conditioned medium (B), or SB623 cellconditioned medium (C).

FIGS. 7A and 7B show measurements of vessel sprouting and branching inan aortic ring assay. FIG. 7A shows counts of new vessels and ofbranchpoints in the new vessels. For each of the three pairs of bars,the left bar shows measurements of new vessel formation and the rightbar shows measurements of vessel branching. The left-most pair of bars(“Control”) shows results obtained from control aortic rings; the centerpair of bars (“MSC CM”) shows results obtained from aortic ringscultured for 10 days in MSC conditioned medium; and the right-most pairof bars (“SB623 CM”) shows results obtained from aoritc rings culturedfor 10 days in SB623 cell conditioned medium.

FIG. 7B show ratios of branchpoints to new vessels for control aorticrings (left bar), rings cultured 10 days in MSC conditioned medium(center bar) and rings cultured 10 days in SB623 cell conditioned medium(right bar).

Values shown are Mean±SEM for 7 donor pairs. “*” indicates p<0.05compared to control group.

FIG. 8 shows levels of four different trophic factors in conditionedmedium from MSCs (light bars) and SB623 cells (dark bars). Proteinlevels are expressed as picograms per ml of conditioned medium per 10⁶cells. Conditioned media from MSCs (and SB623 cells derived therefrom)from four different human donors were tested, as indicated in thefigure. Levels of angiogenin, angiopoietin-2, heparin-binding epidermalgrowth factor-like growth factor (HB-EGF), and placental growth factor(PIGF) are shown.

FIG. 9 shows levels of ten different cytokines in conditioned mediumfrom MSCs and SB623 cells. Cells for production of conditioned mediumwere obtained from four different donors (D1, D2, D3 and D4), asindicated in the figure. This figure highlights the vast amounts of VEGFproduced by MSCs and SB623 cells, compared to the levels of the othertrophic factors tested. Abbreviations are given in the legend to Table 1(Example 6).

FIGS. 10A and 10B show the effects of a VEGF receptor inhibitor onimprovements in HLTVEC viability promoted by SB623 cell-conditionedmedium. FIG. 10A shows the fraction of cells permeable to propidiumiodide in cultures of HUVECs that had been starved for serum and growthfactors. Left-most bar shows results obtained from control serum/growthfactor-starved HUVECs; center bar shows results for serum/growthfactor-starved HUVECs cultured for five days in the presence ofconditioned medium from SB623 cells, and the right-most bar showsresults for sertunlgrowth factor-starved HUVECs cultured for five daysin the presence of conditioned medium from SB623 cells and 50 nM SU5416.Results were averaged from two donors; “*” indicates p<0.05 with respectto control cultures; “#” indicates p<0.05 with respect to culturesexposed to SB623 cell conditioned medium and SU5416.

FIG. 10B shows measurement of the fraction of cells expressing Bcl-2 ina culture of HUVECs that had been starved for serum and growth factors.Left-most bar shows results obtained from control serum/growthfactor-starved HUVECs; center bar shows results for serum/growthfactor-starved HUVECs cultured for five days in the presence ofconditioned medium from SB623 cells, and the right-most bar showsresults for serum/growth factor-starved HUVECs cultured for five days inthe presence of conditioned medium from SB623 cells and 50 nM SU5416.Results, averaged from duplicate donors, were obtained by measuringfluorescence of cells stained with a fluorescein-conjugated anti-Bcl-2antibody and subtracting fluorescence of cells exposed tofluorescein-conjugated IgG.

FIG. 11 shows measurement of the fraction of cells expressing Ki67 inHUVEC cultures exposed to SB623 cell conditioned medium in the presenceand absence of the VEGFR2 inhibitor SU5416, and by control cellscultured in the absence of CM. The left-most (clear) bar shows resultsobtained from control serum/growth factor-starved HUVECs; the center(black) bar shows results for serum/growth factor-starved HUS ECscultured in the presence of conditioned medium from SB623 cells, and theright-most (gray) bar shows results for serumlgrowth factor-starvedHUVECs cultured in the presence of conditioned medium from SB623 cellsand 50 nM SU5416. Values shown are mean+SEM for two separate donors ofSB623 cells. “*” indicates p<0.05 with respect to the negative controlcultures (no conditioned medium); “#” indicates p<0.05 with respect toSU5416-treated cultures.

FIG. 12 shows the effects of a VEGF receptor inhibitor on theenhancement of tube formation by HUVECs promoted by MSC- and SB623cell-conditioned media. The top row shows cells cultured in the absenceof the inhibitor. The left-most panel of the top row (“neg”) shows aphase-contrast photomicrograph of control HUVECs following culture for16 hours in Opti-MEM Medium. The second panel from the left (“+10 ngVEGF”) shows a phase-contrast photomicrograph of HUVECs followingculture for 16 hours in Opti-MEM Medium to which 10 ng/ml VEGF wasadded. The third panel from the left. (“+MSC-CM”) shows a phase-contrastphotomicrograph of HUVECs following culture for 16 hours inMSC-conditioned medium. The rightmost panel (“+SB623-CM”) shows aphase-contrast photomicrograph of HUVECs following culture for 16 hoursin SB623 cell-conditioned medium. Panels in the bottom row showphotomicrographs of HUVECs under the same conditions as in the top rowbut with the addition of 50 nM SU5416.

FIG. 13 shows quantitation of tube formation by HUVECs exposed to SB623cell conditioned medium in the presence and absence of the VEGFR2inhibitor SU5416, and by control cells cultured in the absence of CM.

For each time point, the left-most (clear) bar shows results obtainedfrom control serum/growth factor-starved HUVECs; the center (black) barshows results for serum/growth factor-starved HUVECs cultured in thepresence of conditioned medium from SB623 cells, and the right-most(gray) bar shows results for serum/growth factor-starved HUVECs culturedin the presence of conditioned medium from SB623 cells and 50 nM SU5416.Values shown are mean+SEM for three separate donors of SB623 cells. “*”indicates p<0.05 with respect to the negative control cultures (noconditioned medium); “#” indicates p<0.05 with respect to SU5416-treatedcultures.

FIG. 14 shows the effects of a VEGF receptor inhibitor on enhancement ofvessel outgrowth and branching promoted by SB623 cell-conditioned mediumin an aortic ring assay. In the upper row, the left panel shows aphotomicrograph of an aortic ring after culture for 10 days on aRGF-basement gel in OptiMEM medium (“Negative control”). The centerpanel shows a photomicrograph of an aortic ring after culture for 10days in SB623 cell conditioned medium (“SB623-CM”). The right panelshows a photomicrograph of an aortic ring after culture for 10 days inSB623 cell conditioned medium containing 50 nM SU5416(“+SB623-CM+SU5416”). Enlargements of certain regions of eachphotomicrograph are shown in the lower row.

DETAILED DESCRIPTION

Disclosed herein are new methods and compositions for modulation ofangiogenesis. In particular, factors secreted by SB623 cells (cellsdescended from MSCs that have been transfected with a vector containingsequences encoding a Notch intracellular domain) promote survival andproliferation of endothelial cells in vitro under serum- and growthfactor-deprived conditions, and stimulate vascular tube formation byhuman umbilical vein endothelial cells. In addition, conditioned mediumfrom SB623 cells promoted endothelial sprouting and branching in arodent aortic ring assay.

Practice of the present disclosure employs, unless otherwise indicated,standard methods and conventional techniques in the fields of cellbiology, toxicology, molecular biology, biochemistry, cell culture,immunology, oncology, recombinant DNA and related fields as are withinthe skill of the art. Such techniques are described in the literatureand thereby available to those of skill in the art. See, for example,Alberts, B. et al., “Molecular Biology of the Cell,” 5^(th) edition,Garland Science, New York, N.Y., 2008; Voet, D. et al. “Fundamentals ofBiochemistry: Life at the Molecular Level,” 3^(rd) edition, John Wiley &Sons, Hoboken, N.J., 2008; Sambrook, J. et al., “Molecular Cloning: ALaboratory Manual,” 3^(rd) edition, Cold Spring Harbor Laboratory Press,2001; Ausubel, F. et al., “Current Protocols in Molecular Biology,” JohnWiley & Sons New York, 1987 and periodic updates; Freshney, R. I.,“Culture of Animal Cells: A Manual of Basic Technique,” 4^(th) edition,John Wiley & Sons, Somerset, N.J., 2000; and the series “Methods inEnzymology,” Academic Press, San Diego, Calif.

For the purposes of the present. disclosure, “angiogenesis” refers tothe formation of new vasculature (e.g., blood vessels; e.g., veins,arteries, venules arterioles, capillaries). Angiogenesis can occur bysprouting of new vessels from an existing vessel, and/or by branching ofa vessel. Angiogenesis also includes the attendant processes of matrixremodeling and cell recruitment (e.g., recruitment of smooth musclecells, monocytes and/or pericytes).

“MSCs” refer to adherent, non-heniatopoietic stem cells obtained frombone marrow. These cells are variously known as mesenchymal stem cells,mesenchymal stromal cells, marrow adherent stromal cells, marrowadherent stem cells and bone marrow stromal cells.

Stroke

“Stroke” is the name given to conditions resulting from impairment ofblood flow in the brain. Such cerebrovascular impairment can result, forexample, from intracranial hemorrhage, or from reduction or blockage ofblood flow in the brain (i.e., cerebral ischemia). Ischemic blockagescan result from thrombosis (i.e., formation of a clot in situ in acranial vessel or a vessel supplying the brain) or from a cerebralembolism migration of a clot to a site in the brain). The damageresulting from ischemic or hemorrhagic stroke usually results inimpairment of neurological function. Additional information relating todifferent types of stroke, and their characteristics, is found inco-owned U.S. Pat. No. 8,092,792; the disclosure of which isincorporated by reference in its entirety herein for the purpose ofdescribing different types of stroke and their characteristics.

Mesenchymal Stem Cells (MSCs)

The present disclosure provides methods for promoting angiogenesis bytransplanting SB623 cells to a site of ischemic injury in a subject.SB623 cells are obtained from marrow adherent stromal cells, also knownas mesenchymal stem cells (MSCs), by expressing the intracellular domainof the Notch protein in the MSCs. MSCs are obtained by selectingadherent cells (i.e., cells that adhere to tissue culture plastic) frombone marrow.

Exemplary disclosures of MSCs are provided in U.S. patent applicationpublication No. 2003/0003090; Prockop (1997) Science 276:71-74 and Jiang(2002) Nature 418:41-49. Methods for the isolation and purification ofMSCs can be found, for example, in U.S. Pat. No. 5,486,359; Pittenger etal. (1999) Science 284:143-147 and Dezawa et al. (2001) Eur. J.Neurosci. 14:1771-1776. Human MSCs are commercially available (e.g.,BioWhittaker, Walkersville, Md.) or can be obtained from donors by,e.g., bone marrow aspiration, followed by selection for adherent bonemarrow cells. See, e.g., WO 2005/100552.

MSCs can also be isolated from umbilical cord blood. See, for example,Campagnoli et al. (2001) Blood 98:2396-2402; Erices et al. (2000) Br. J.Haematol. 109:235-242 and Hou et al. (2003) Int. Hematol. 78:256-261.

Notch Intracellular Domain

The Notch protein is a transmembrane receptor, found in all metazoans,that influences cell differentiation through intracellular signaling.Contact of the Notch extracellular domain with a Notch ligand (e.g.,Delta, Serrate, Jagged) results in two proteolytic cleavages of theNotch protein, the second of which is catalyzed by a γ-secretase andreleases the Notch intracellular domain (NICD) into the cytoplasm. Inthe mouse Notch protein, this cleavage occurs between amino acidsgly1743 and val1744. The NICD translocates to the nucleus, where it actsas a transcription factor, recruiting additional transcriptionalregulatory proteins (e.g., MAM, histone acetylases) to relievetranscriptional repression of various target genes (e.g., Hes 1).

Additional details and information regarding Notch signaling are found,for example in Artavanis-Tsakonas et al. (1995) Science 268:225-232;Mumm and Kopan (2000) Develop. Biol. 228:151-165 and Ehebauer et al.(2006) Sci. STKE 2006 (364), cm7. [DOI: 10.1126/stke.3642006cm7].

Cell Culture and Transfection

Standard methods for cell culture are known in the art. See, forexample, R. I. Freshney “Culture of Animal Cells: A Manual of BasicTechnique,” Fifth Edition, Wiley, New York, 2005.

Methods for introduction of exogenous DNA into cells (i.e.,transfection) are also well-known in the art. See, for example, Sambrooket al “Molecular Cloning: A Laboratory Manual,” Third Edition, ColdSpring Harbor Laboratory Press, 2001; Ausubel et al., “Current Protocolsin Molecular Biology,” John Wiley & Sons, New York, 1987 and periodicupdates.

SB623 Cells

In one embodiment for the preparation of SB623 cells, a culture of MSCsis contacted with a polynucleotide comprising sequences encoding a Notchintracellular domain (MCD): e.g., by transfection; followed byenrichment of transfected cells by drug selection and further culture.See, for example, U.S. Pat. No. 7,682,825 (Mar. 23, 2010); U.S. PatentApplication Publication No. 2010/0266554 (Oct. 21, 2010); and WO2009/023251 (Feb. 19, 2009); all of whiCh disclosures are incorporatedby reference, in their entireties, for the purposes of describingisolation of mesenchymal stem cells and conversion of mesenchymal stemcells to SB623 cells (denoted “neural precursor cells” and “neuralregenerating cells” in those documents).

In these methods, any polynucleotide encoding a Notch intracellulardomain e.g., vector) can be used, and any method for the selection andenrichment of transfected cells can be used. For example, in certainembodiments, MSCs are transfected with a vector containing sequencesencoding a Notch intracellular domain and also containing sequencesencoding a drug resistance marker (e.g. resistance to G418). Inadditional embodiments, two vectors, one containing sequences encoding aNotch intracellular domain and the other containing sequences encoding adrug resistance marker, are used for transfection of MSCs. In theseembodiments, selection is achieved, after transfection of a cell culturewith the vector or vectors, by adding a selective agent (e.g., G418) tothe cell culture in an amount sufficient to kill cells that do notcomprise the vector but spare cells that do. Absence of selectionentails removal of said selective agent or reduction of itsconcentration to a level that does not kill cells that do not comprisethe vector. Following selection e.g., for seven days) the selectiveagent is removed and the cells are further cultured (e.g., for twopassages).

Preparation of SB623 cells thus involves transient expression of anexogenous Notch intracellular domain in a MSC. To this end, MSCs can betransfected with a vector comprising sequences encoding a Notchintracellular domain wherein said sequences do not encode a full-lengthNotch protein. All such sequences are well known and readily availableto those of skill in the art. For example, Del Amo et al. (1993)Genomics 15:259-264 present the complete amino acid sequences of themouse Notch protein; while Mumm and Kopan (2000) Devel. Biol.228:151-165 provide the amino acid sequence, from mouse Notch protein,surrounding the so-called S3 cleavage site which releases theintracellular domain. Taken together, these references provide theskilled artisan with each and every peptide containing a Notchintracellular domain that is not the full-length Notch protein; therebyalso providing the skilled artisan with every polynucleotide comprisingsequences encoding a Notch intracellular domain that does not encode afull-length Notch protein. The foregoing documents (Del Amo and Mumm)are incorporated by reference in their entireties for the purpose ofdisclosing the amino acid sequence of the full-length Notch protein andthe amino acid sequence of the Notch intracellular domain, respectively.

Similar information is available for Notch proteins and nucleic acidsfrom additional species, including rat, Xenopus, Drosophila and human.See, for example. Weinmaster et al. (1991) Development 113:199-205;Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No.NM_017167 (and references cited therein); SwissProt P46531 (andreferences cited therein): SwissProt. Q01705 (and references citedtherein); and GenBank CAB40733 (and references cited therein). Theforegoing references are incorporated by reference in their entiretiesfor the purpose of disclosing the amino acid sequence of the full-lengthNotch protein and the amino acid sequence of the Notch intracellulardomain in a number of different species.

In additional embodiments, SB623 cells are prepared by introducing, intoMSCs, a nucleic acid comprising sequences encoding a Notch intracellulardomain such that the MSCs do not express exogenous Notch extracellulardomain. Such can be accomplished, for example, by transfecting MSCs witha vector comprising sequences encoding a Notch intracellular domainwherein said sequences do not encode a full-length Notch protein.

Additional details on the preparation of SB623 cells, and methods formaking cells with properties similar to those of SB623 cells which canbe used in the methods disclosed herein, are found in U.S. Pat. No.7,682,825; and U.S. Patent Application

Publication Nos. 2010/0266554 (Oct. 21, 2010) and 2011/0229442 (Sep. 22,2011); the disclosures of which are incorporated by reference herein forthe purposes of providing additional details on, and alternative methodsfor the preparation of, SB623 cells, and for providing methods formaking cells with properties similar to those of SB623 cells. See alsoDezawa et al. (2004) J. Clin. Invest. 113:1701-1710.

Uses

As disclosed herein, the inventors have discovered that descendants ofmesenchymal stem cells in which a Notch intracellular domain has beentransiently expressed (i.e., SB623 cells) have angiogenic activity; andthat said cells synthesize and secrete angiogenic factors. Accordingly,transplantation of SB623 cells is useful for treatment of disorders inwhich a therapeutic benefit can be achieved by increasing angiogenesisin a subject. Such disorders include, but are not limited to, cerebralischemia (e.g., stroke), cardiac ischemia ischemic heart disease),ischemia of the bowel ischemic colitis, mesenteric ischemia), ischemiaof the limb, cutaneous ischemia, ocular ischemic syndrome (e.g., retinalischemia) and cerebral palsy.

Thus, SB623 cells as described herein can be used in a number of methodsrelated to stimulation of angiogenesis. These include, but are notlimited to, treatment of any of the disorders mentioned in the previousparagraph, augmentation of angiogenesis, repair of ischemic damage,preventing death of endothelial cells, enhancing survival of endothelialcells, stimulating proliferation of endothelial cells, and/or enhancingthe branching of blood vessels.

Such methods can be performed in vitro or in a subject. The subject canbe a mammal, preferably a human. Stimulation of angiogenesis by SB623cells, and the attendant effects of such stimulation as disclosedherein, can occur, for example, in the central nervous system (e.g., inthe brain).

Transplantation of SB623 cells can also be used in methods for providingone or more angiogenic trophic factors to a subject. Such factorsinclude, but are not limited to, angiogenin, angiopoietin-2, epidermalgrowth factor, basic fibroblast growth factor, heparin-bindingepithelial growth factor-like growth factor, hepatocyte growth factor,leptin platelet-derived growth factor-BB, placental growth factor andvascular endothelial growth factor.

In additional embodiments, SB623 cells can be used in combination with asecond pro-angiogenic agent, in combination therapies for increasingangiogenesis in a subject. Said combination therapies can be used forall of the purposes set forth above. The second pro-angiogenic agent canbe, e.g., a small molecule drug, a nucleic acid or a polypeptide (e.g.,antibody, transcription factor). Exemplary nucleic acids aretriplex-forming nucleic acids, ribozymes and siRNAs that activateexpression of angiogenic proteins andlor block expression ofanti-angiogenic proteins. Exemplary antibodies are those that bind toandlor inhibit the activity of angiogenic proteins (or other angiogenicagents). Exemplary transcription factors are those that inhibittranscription of a gene encoding one or more anti-angiogenic protein(s),as well as those that activate the transcription of one or morepro-angiogenic protein(s). Anti-angiogenic and pro-angiogenic proteinsare known in the art. Exemplary anti-angiogenic proteins include pigmentepithelium derived factor (PERF) and placental growth factor (PlGF).Exemplary pro-angiogenic proteins include vascular endothelial growthfactor (VEGF) angiopoietin, and hepatocyte growth factor (HGF).

In certain embodiments, transcription factors as disclosed above arenon-naturally-occurring engineered) transcription factors. An example ofsuch a non-naturally-occurring transcription factor is anon-naturally-occurring zinc finger protein that has been engineered tobind to a DNA sequence in cellular chromatin that regulatestranscription of a target gene (e.g., a VEGF gene). Said engineered zincfinger transcription factors comprise, in addition to an engineered zincfinger DNA-binding domain, a transcriptional regulatory domain (e.g., atranscriptional activation domain or a transcriptional repressiondomain), as are known in the art.

Methods for engineering zinc finger DNA binding domains, to bind to aDNA sequence of choice, are well-known in the art. See, for example,Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pala et al. (2001)Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol.19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Chooet al. (2000) Curr. Opin. Struct. Biol. 10:411-416. Zinc finger bindingdomain are engineered to have a novel binding specificity, compared to anaturally-occurring zinc finger protein. Engineering methods include,but are not limited to, rational design and various types of empiricalselection methods. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, U.S. Pat. Nos. 6,140,081;6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617; U.S.Patent Application. Publication Nos. 2002/0165356; 2004/0197892;2007/0154989; 2007/0213269; and International Patent ApplicationPublication Nos. WO 98/53059 and WO 2003/016496.

Exemplary selection methods, including phage display, interaction trap,hybrid selection and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,140,466; 6,200,759;6,242,568; 6,410,248; 6,733,970; 6,790,941; 7,029,847 and 7,297,491; aswell as U.S. Patent Application Publication Nos. 2007/0009948 and2007/0009962; WO 98/37186; WO 01/60970 and GB 2,338,237.

Enhancement of binding specificity for zinc forger binding domains hasbeen described, for example, in U.S. Pat. No. 6,794,136 (Sep. 21, 2004).Additional aspects of zinc finger engineering, with respect tointer-finger linker sequences, are disclosed in U.S. Pat. No. 6,479,626and U.S. Patent Application Publication No. 2003/0119023. See also Mooreet al. (2001a) Proc. Natl. Acad. Sci. USA 98:1432-1436; Moore et al.(200b) Proc. Natl. Acad. Sci. USA 98:1437-1441 and WO 01/53480.

Transcriptional activation and repression domain are known in the art.See, e.g., Science 269:630 (1995). Exemplary transcriptional activationdomains include p65, VP16 and VP64. Exemplary transcriptional repressiondomains include KRAB, KAP-1, MAD, FKHR, ERD and SID. Functional domainsfrom nuclear hormone receptors can act as either activators orrepressors, depending upon the presence of a ligand. See also U.S. Pat.No. 7,985,887.

Formulations, Kits and Routes of Administration

Therapeutic compositions comprising SB623 cells as disclosed herein arealso provided. Such compositions typically comprise the SB623 cells anda pharmaceutically acceptable carrier. Supplementary active compoundscan also be incorporated into SB623 cell compositions (see below).

The therapeutic compositions disclosed herein are useful for, interalia, stimulating angiogenesis after occurrence of a stroke or otherischemic injury in a subject. Accordingly, a “therapeutically effectiveamount” of a composition comprising SB623 cells can be any amount thatstimulates angiogenesis. For example, dosage amounts can vary from about100; 500; 1,000; 2,500; 5,000; 10,000; 20,000; 50,000; 100,000; 500,000;1,000,000; 5,000,000 to 10,000,000 cells or more (or any integral valuetherebetween); with a frequency of administration of, e.g., once perday, twice per week, once per week, twice per month, once per month,depending upon, e.g., body weight, route of administration, severity ofdisease, etc.

Various pharmaceutical compositions and techniques for their preparationand use are known to those of skill in the art in light of the presentdisclosure. For a detailed listing of suitable pharmacologicalcompositions and techniques for their administration one may refer totexts such as Remington's Pharmaceutical Sciences, 17th ed. 1985;Brunton et al., “Goodman and Gilman's The Pharmacological Basis ofTherapeutics,” McGraw-Hill, 2005 University of the Sciences in.Philadelphia (eds.) “Remington: The Science and Practice of Pharmacy,”Lippincott Williams & Wilkins, 2005: and University of the Sciences inPhiladelphia (eds.), “Remington: The Principles of Pharmacy Practice,”Lippincott Williams & Wilkins, 2008.

The cells described herein can be suspended in a physiologicallycompatible carrier for transplantation. As used herein, the term“physiologically compatible carrier” refers to a carrier that iscompatible with the SB623 cells and with any other ingredients of theformulation, and is not deleterious to the recipient thereof. Those ofskill in the art are familiar with physiologically compatible carriers.Examples of suitable carriers include cell culture medium (e.g., Eagle'sminimal essential medium), phosphate buffered saline, Hank's balancedsalt solution+/−glucose (HBSS), and multiple electrolyte solutions suchas Plasma-Lyte™ A (Baxter).

The volume of a SB623 cell suspension administered to a patient willvary depending on the site of implantation, treatment goal and number ofcells in solution. Typically the amount of cells administered to apatient will be a therapeutically effective amount. As used herein, a“therapeutically effective amount” or “effective amount” refers to thenumber of transplanted cells which are required to effect treatment ofthe particular disorder; i.e., to produce a reduction in the amountand/or severity of the symptoms associated with that disorder. Forexample, in the case of stroke, transplantation of a therapeuticallyeffective amount of SB623 cells results in new vessel growth, vesselsprouting and vessel branching, e.g., in an area that has been damagedby ischemic. Therapeutically effective amounts vary with the type andextent of ischemic damage, and can also vary depending on the overallcondition of the subject.

The disclosed therapeutic compositions can also include pharmaceuticallyacceptable materials, compositions or vehicles, such as a liquid orsolid filler, diluent, excipient, solvent or encapsulating material,i.e., carriers. These carriers can, for example, stabilize the SB623cells and/or facilitate the survival of the SP623 cells in the body.Each carrier should be “acceptable” in the sense of being compatiblewith the other, ingredients of the formulation and not injurious to thesubject. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations. Wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

Exemplary formulations include, but are not limited to, those suitablefor parenteral administration, e.g., intrapuhnonary, intravenous,intra-arterial, intra-ocular, intra-cranial, sub-meningial, orsubcutaneous administration, including formulations encapsulated inmicelles, liposomes or drug-release capsules (active agents incorporatedwithin a biocompatible coating designed for slow-release); ingestibleformulations; formulations for topical use, such as eye drops, creams,ointments and gels; and other formulations such as inhalants, aerosolsand sprays. The dosage of the compositions of the disclosure will varyaccording to the extent and severity of the need for treatment, theactivity of the administered composition, the general health of thesubject, and other considerations well known to the skilled artisan.

In additional embodiments, the compositions described herein aredelivered locally to a site of ischemic damage. Localized deliveryallows for the delivery of the composition non-systemically, therebyreducing the body burden of the composition as compared to systemicdelivery. Such local delivery can be achieved, for example, byintra-cranial injection, or through the use of various medicallyimplanted devices including, but not limited to, stems and catheters, orcan be achieved by inhalation, phlebotomy, or surgery. Methods forcoating, implanting, embedding, and otherwise attaching desired agentsto medical devices such as stents and catheters are established in theart and contemplated herein.

Another aspect of the present disclosure relates to kits for carryingout the administration of SB623 cells, optionally in combination withanother therapeutic agent, to a subject. In one embodiment, a kitcomprises a composition of SB623 cells, formulated in a pharmaceuticalcarrier, optionally containing, e.g., a pro-angiogenic agent (seebelow), formulated as appropriate, in one or more separatepharmaceutical preparations.

Combination Therapies

In certain embodiments, SB623 cell compositions can be used incombination with other compositions comprising substances that stimulateangiogenesis (“pro-angiogenic agents”), e.g., for treatment of stroke.The compositions can be administered sequentially in any order orconcurrently. Accordingly, therapeutic compositions as disclosed hereincan contain both SB623 cells and a pro-angiogenic agent. In additionalembodiments, separate therapeutic compositions, one comprising SB623cells and the other comprising a pro-angiogenic agent, can beadministered to the subject, either separately or together.

In certain embodiments, a pro-angiogenic agent is a protein (e.g.,fibroblast growth factor, platelet-derived growth factor, transforminggrowth factor alpha, hepatocyte growth factor, vascular endothelialgrowth factor, sonic hedgehog, MAGP-2, HIF-1, PR-39, RTEF-1, c-Myc,TFII, Egr-1, ETS-1) or a nucleic acid encoding such a protein. See, forexample, Vincent et al. (2007) Gene Therapy 14:781-789. In otherembodiments, a pro-angiogenic agent can be a small. RNA molecule (e.g.,siRNA, shRNA, microRNA) or a ribozyme that targets a nucleic acidencoding an inhibitor of angiogenesis. In additional embodiments, apro-angiogenic agent can be a triplex-forming nucleic acid that binds toDNA sequences regulating the expression of a protein that inhibitsangiogenesis, such as to block transcription of the gene encoding theprotein.

In additional embodiments, a pro-angiogenic agent is a transcriptionfactor that activates expression of a pro-angiogenic molecule (e.g.,protein). Naturally-occurring transcription factors (such as, forexample, HIF-1alpha) that regulate the expression of pro-angiogenicproteins, are known. In addition, synthetic transcriptional regulatoryproteins can be constructed by genetic engineering. For example, methodsfor the design of zinc finger DNA-binding domains that bind to asequence of interest, and methods for the fusion of such zinc fingerDNA-binding domains to transcriptional activation and repressiondomains, have been described. See, for example, U.S. Pat. Nos.6,534,261: 6,607,882; 6,785,613; 6,794,136; 6,824,978; 6,933,16,979,539; 7,013,219: 7,177,766; 7,220,719; and 7,788,044. These methodscan be used to synthesize non-naturally-occurring proteins that activatetranscription of any gene encoding a pro-angiogenic protein. hiaddition, synthetic zinc finger transcriptional activators of thevascular endothelial growth factor (VEGF) gene have been described. See,e.g., U.S. Pat. Nos. 7,026,462; 7,067,317; 7,560,440: 7,605,140; and8,071,564. Accordingly, a non-naturally-occurring (i.e., synthetic) zincfinger protein that activates transcription of the VEGF gene can beused, in combination with SB623 cells, for augmenting angiogenesis,e.g., in the treatment of stroke.

In additional embodiments, a natural or synthetic transcriptionalregulatory protein (e.g., a synthetic zinc finger transcriptionalregulatory protein) that inhibits transcription of an anti-angiogenicmolecule can be used as a pro-angiogenic agent.

EXAMPLES Example 1 Conditioned Medium

MSCs and SB623 cells were obtained and/or prepared as described. See,for example, U.S. Pat. No. 7,682,825 (Mar. 23, 2010) and U.S. PatentApplication Publications Nos. 2010/0266554 (Oct. 21, 2010), 201010310529(Dec. 9, 2010), 2011./02.29442 (Sep. 22., 2011), and 201110306137 (Dec.15, 2011); the disclosures of which are incorporated by reference intheir entireties for the purposes of describing the preparation of SB623cells (variously referred to as “neural precursor cells” and “neuralregenerating cells” in those documents). Cells were cultured in growthmedium, which contained alpha-MEM (Mediatech, Herndon, Va.) supplementedwith 10% fetal bovine serum (FBS, Hyclone, Logan, Utah), 2 mML-glutamine and 1% penicillin/streptomycin (both from Invitrogen,Carlsbad, Calif.). MSCs and SB623 cells typically expressed CD29, CD90and CD105; and did not express CD31, CD34, or CD45, as determined byflow cytometry.

For use in the experiments described herein, frozen MSCs and SB623 cellsfrom the same human donor were thawed, re-plated in growth medium, andallowed to recover for approximately one week. To obtain conditionedmedium, cells were grown to approximately 90% confluence (˜45,000cells:cm), the plates were rinsed once with phosphate buffered saline(PBS) and the medium was then replaced with OptiMEM® medium (Invitrogen,Carlsbad, Calif.), maintaining the same cell density. Conditioned mediumwas collected 72 hours later. Frozen samples of conditioned medium wereslowly warmed to 37° C. prior to use.

Example 2 Effect of SB623 Cell-secreted Factors on HUVEC Survival

Cerebral ischemia can result in loss of nutrient supply to the affectedarea. To determine if soluble factors from SB623 cells and MSCs haverestorative effects on nutrient-deprived endothelial cells, humanumbilical vein endothelial cells (HUVECs) were cultured in mediumdepleted of serum and growth factors for 24 hours, then exposed toconditioned medium (CM) from MSCs or SB623 cells. Control culturesremained in serum- and growth factor-depleted medium without addition ofCM. Viability and proliferative capacity of the HUVECs were thenassessed.

For these experiments, human umbilical vein endothelial cells werepassed twice, then 7.5×10⁵ cells were plated in EBM-2/EGGS medium(Endothelial Basal Medium-2/Endothelial Cell Growth Supplements; Lonza,Walkersville, Md.) on T-75 flaSks coated with 0.1% gelatin and culturedfor 24 hours. The FILIVEC monolayers were rinsed twice with warns PBSand incubated with 12 ml of fresh EBM-2 medium overnight at 37° C., 5%CO₂. Effects of CM were then assessed by withdrawing 6 ml of medium fromeach flask, and replacing it with 6 ml fresh OptiMEM (control), 6 ml MSCconditioned medium, or 6 ml SB623 cell conditioned medium (conditionedmedia prepared as described in Example 1). After 7 days, non-adherentand adherent cells were collected, centrifuged at 1400 rpm for 5 min,and divided into three fractions for subsequent staining analyses (PI,Bcl-2 and Ki67).

To quantify cell death, cells were stained with propidium iodide (PI),since dead cells are permeable to PI. Cells were stained with 5 ug/ml PIfor 30 min at room temperature, and, flow cytometry acquisition andanalysis were conducted using the FL-2 logarithmic channel of a BDFACSCalibur CellQuest program (BD Biosciences, San Jose, Calif.). Forthis assay, 3 different human donor pairs were tested. The results areshown in FIG. 1. In control HUVEC cultures maintained innutrient-deprived medium for 7 days, more than 70% of the cells werepositive for propidium iodide staining. Addition of either SB623- orMSC-conditioned medium significantly reduced the percentage of propidiumiodide positive cells (p<0.05).

These results indicate that both MSC conditioned medium and SB623 cellconditioned medium significantly reduced death of endothelial cells(i.e., reduced the number of propidium iodide-positive HUVECs) resultingfrom serum and growth factor starvation.

Bcl-2 is an anti-apoptotic protein originally identified as beingoverexpressed in certain B-cell lymphomas. Accordingly, the fraction ofcells expressing the Bcl-2 protein was measured in serumigrowthfactor-starved HUVECs as an indicator of their apoptotic potential. ForBcl-2 measurement, cells were fixed in 4% parafonnaldehyde andpermeabilized with 0.1% Triton-X100 for one hour. Followingpermeabilization, cells were stained for one hour, on ice, withfluorescein-conjugated anti-Bcl-2 antibody, then samples were washed,acquired, and analyzed on the FL-1 channel of a BD FACSCalibur. Cellsexposed to fluorescein-conjugated IgG were used as a negative control.For these assays, 3 different human donor pairs were tested.

The results, shown in FIG. 2, indicate that presence of either MSCconditioned medium or SB623 cell conditioned medium significantlyincreased the fraction of Bcl-2-positive cells in cultures ofserum-starved endothelial cells.

The fact that conditioned medium from MSCs or from SB623 cells decreasedthe number of dead (PI-positive) cells and increased of the number ofcells expressing the anti-apoptotic Bcl-2 protein shows that both MSCsand SB623 cells secrete factors that enhance endothelial cell survival.

Example 3 Effect of SB623 Cell-Secreted Factors on HUVEC Proliferation

Ki67 is a protein present in cells exiting from the G0 (quiescent) phaseof the cell cycle; therefore Ki67 levels can be used as a measure ofcell proliferation. The fraction of cells expressmg Ki67 protein wasmeasured in HUVECs that had been starved for serum and growth factors,then cultured with conditioned medium from either MSCs or SB623 cells.

For Ki67 measurement, HUVECs were cultured and exposed to CM asdescribed in Example 2. Cells were fixed in 4% paraformaldehyde andpermeabilized with 0.1% Triton-X100 for one hour. Followingpermeabilization, cells were stained for one hour on ice withfluorescein-conjugated anti-KI67 antibody, then samples were washed,acquired, and analyzed on the FL-1 channel of a BD FACSCalibur. Cellsexposed to fluorescein-conjugated IgG were used as a negative control.For these assays, 3 different human donor pairs were tested.

FIG. 3 shows that culture of starved HUVECs in the presence ofconditioned medium from either MSCs or SB623 cells resulted in anincreased fraction of cells expressing Ki67, compared to control HUVECsnot exposed to conditioned medium. The fact that conditioned medium fromMSCs or from SB623 cells increased the number of cells expressing theproliferation-associated Ki67 protein shows that both MSCs and SB623cells secrete factors that enhance endothelial cell proliferation.

The results presented in this and the previous example revealedsignificant increases in survival and proliferation of HUVECs when theseendothelial cells were cultured for 7 days with MSC- or SB623cell-conditioned medium, compared to culture in unconditioned medium(p<0.05).

Example 4 Effect of SB623 Cell-Secreted Factors on Tube Formation byEndothelial Cells

A HUVEC tube formation assay was used to test the ability of MSCs andSB623 cells to elaborate factors that stimulate vessel formation. See,for example, E J Smith & C A Staton, “Tubule formation assays,” inAngiogenesis Assays—A Critical Appraisal of Current Techniques, (Staton,Lewis & Bicknell, eds.). John Wiley & Sons, Ltd., West Sussex, UK., pp.65-87, 2006; and Goodwin (2007) Microvasc. Res. 74:172-183.

HUVECs were passed five times in E8M-2/ECGS medium, then transferred toalpha-MEM/0.5%1 BS/2 mM glutamine/pen-strep, at a density of 1×10⁵ cellsml. After 24 hours, IITIVECs were harvested using 0.25% trypsin-EDTA,rinsed, and resuspended in α-MEMS2mM glutamine/pen-strep at a density of1×10⁵ cells/ml. A mixture of 75 ul of HUVECs plus 75 ul of either MSC-or SB623-conditioned medium (Example 1), or 75 ul OptiMEM medium as anegative control, was added to each well of a 96-well plate that hadbeen pre-treated by adding 50 ul of Reduced Growth Factor (RGF)-basementgel (Invitrogen, Carslsbad, Calif.) per well and incubating the platesat 37° C. for 45 minutes. For this assay, MSCs were obtained from 3different human donors, and a portion of the MSCs from each donor wereconverted to SB623 cells.

After 16 hours, the cultures were examined by phase contrast microscopyand photographed. The number of complete tubes (formed by contiguouscells) was quantified by an experimenter blinded to the group. Aphotograph showing results from one of the three donors is shown in FIG.4. The results of assays using MSCs and SB623 cells from all threedonors, summarized in FIG. 5, indicate that tube formation is stronglyenhanced by conditioned medium from either MSCs or SB623 cells. Thus,MSCs and SB623 cells secrete factors that promote vasculogenesis.

Example 5 Effect of SB623 Cell-Secreted Factors on Vessel Outgrowth andBranching

Restoration of vasculature after ischemic injury requires that survivingendothelial cells receive signals that prompt their migration andinvasion. Such signals may arise from vascular smooth muscle cells,monocytes, and/or macrophages, among others. To test for secretion offactors involved in vessel sprouting and branching, the aortic ringassay was used. See, for example, Nicosia & Ottinetti (1990) Lab.Invest. 63:115-122 and Nicosia (2009) J. Cell. Mol. Med. 13:4113-4136.

For preparation of aortic rings, adult Sprague-Dawley rats wereeuthanized prior to dissection. After clamping off its two ends, theaorta was removed and placed in ice-cold α-MEM/pen-strep medium prior toremoval of the external adipose layer. Adipose-free aorta was rinsedtwice with ice-cold EBM-2/pen-strep medium before being sectioned intorings of 1.0 mm thickness. The aortic rings were then transferred toplates containing EBM-2/pen-strep medium and incubated at 37° C., 5% CO₂for 6 days, with the medium replaced with fresh EBM-2/pen-strep mediumon day 3, to deplete any endogenous rat angiogenic factors. At thatpoint, the medium was replaced with alpha-MEM/pen-strep medium andculture was continued for 24 hours.

On day 0 of the aortic ring assay (seven days after beginning ofculture), 50 μl of reduced growth factor (RGF) basement gel wasdeposited per well of a 24-well plate. An individual aortic ring wasplaced in the middle of each gel-coated well and overlaid with anadditional 25 μl of RGF-basement gel. After allowing 30 minutes at 37°C./5% CO₂ for solidification of the gel, 500 μl of α-MEM/2 mMglutamine/pen-strep was added to each well and incubation was continuedfor an additional 30 minutes. Then, 500 ul of either MSC- orSB623-derived conditioned medium (Example 1) was added. As a negativecontrol, 500 μl of OptiMEM medium was used in place of conditionedmedium.

To assess the angiogenic activity of MSC- and SB623-derived factors,phase contrast photographs were taken on Day 10, and results werequantified by an experimenter blinded to the goup, by counting vesseloutgrowth and branching. Growth of new vessels was quantitated bymeasuring the number of vessels growing out from the ring; and vesselbranching was quantitated by measuring the number of branchpointspresent in vessels growing out from the aortic ring. For this assay, 7different human donor pairs were tested.

Representative results from Day 10 samples are shown in FIG. 6, andresults from seven sets of 10-day cultures are summarized andquantitated in FIG. 7. FIG. 7A shows that conditioned medium from bothMSCs and SB623 cells stimulated an increase in the number ofnewly-sprouted vessels and in the degree of branching, compared tocontrol aortic rings. Moreover, significant increases in vesselbranching were observed in rings cultured in SB623 cell-conditionedmedium (FIG. 6C, FIG. 7A), compared with either rings cultured inMSC-conditioned medium (FIG. 6B, FIG. 7A) or rings cultured inunconditioned medium (FIG. 6A, FIG. 7A). These results indicate thatMSCs and SB623 cells secrete factors that enhance vessel sprouting andvessel branching. In particular, SB623 cells secrete factors thatgreatly enhance vessel branching (see FIG. 7B).

The data presented the foregoing examples indicate that SB623cell-secreted soluble factors promote several aspects of angiogenesis,which contribute to recovery in the injured brain.

Example 6 Statistics

For each experiment (which included 3-4 wells/group), a mean value wasobtained for: (1) the treatment condition for each cell type (eitherMSC- or SB623 cell-derived conditioned medium; one value per human donortested) and (2) the untreated group (one value for each round oftesting). For statistical comparison (SigmaStat, SystatSoftware,Chicago, Ill.) each of these values were used and comparisons were madeusing one way ANOVA between the following groups (1) Control(unconditioned medium: n=3), (2) MSC-conditioned medium (n=3-5): and (3)SB623 cell-conditioned medium (n=3-5). Additional pair-wise comparisonswere made using Tukey's test. An alpha value of 0.05 was used todetermine whether the means were significantly different.

Example 7 Identification of Angiogenic Factors Secreted by MSCs andSB623 Cells

The levels of certain cytokines and trophic factors in conditionedmedium from MSCs and SB623 cells were measured. To obtain conditionedmedium, MSCs or SB623 cells were cultured in growth medium to ˜90%confluence (˜15,000 cells/cm²), at which point medium was removed, thecells were rinsed in PBS, and Opti-MEM® medium (Invitrogen, Carlsbad,Calif.) was added to give a concentration of ˜150,000 cells/ml. Theconditioned medium was collected 72 hours later and assayed using aQuantibody® Human Angiogenesis Array 1 (RayBiotech, Norcross, Ga.)according to the manufacturer's instructions. For each source of MSCs aportion of the cells were cultured directly as MSCs and a portion wereconverted to SB623 cells. Thus, a culture of MSCs from a particulardonor and a culture of SB623 cells made from those MSCs, are referred toas a matched “donor pair.” In this experiment, four donor pairs wereassayed. Results, expressed as protein concentration, were normalized tothe number of cells present in the culture when the conditioned mediumwas collected. FIG. 8 shows results, by donor, for angiogenin, ANG-2,HB-EGF and PIGF. FIG. 9 shows results for these four factors, and sixothers, also by donor, and highlights the large amounts of VEGF producedby MSCs and SB623 cells.

Table 1 shows protein levels averaged among the four donor pairs for theten factors tested. Although levels of trophic factors secreted werevariable among the different donors (as shown, for example, in FIGS. 8and 9), levels of four of the factors (angiogenin, angiopoietin-2,HB-EGF and PIGF) were consistently different between MSCs and SB623cells. Angiogenin, ANG-2 and HB-EGF were more highly expressed by SB623cells, while higher concentrations of PIGF were produced by MSCs.

TABLE 1 Levels of Angiogenic trophic factors in conditioned medium fromMSCs and SB623 Cells MSCs SB623 Cells FACTOR AVG SD AVG SD Angiogenin741 178 985 271 ANG-2 540 252 641 275 EGF — n/a — n/a bFGF 53 7 40 17HB-EGF 205 162 282 228 HGF 123 55 143 75 Leptin 397 226 437 213 PDGF-BB18 22 16 18 PIGF 300 178 171 85 VEGF 30,503 9229 38,119 8692Abbreviations are as follows. ANG-2: angiopoietin-2; EGF: epidermalgrowth factor; bFGF: basic fibroblast growth factor/fibroblast growthfactor 2; HB-EGF: heparin-binding epidermal growth factor-like growthfactor; HGF: hepatocyte growth factor; PDGF-BB: platelet-derived growthfactor-BB; PIGF: placental growth factor; VEGF: vascular endothelialgrowth factor. Numbers refer to cytokine levels expressed as pg/ml/10⁶cells. “AVG” refers to the average value from 4 sources of MSCs and 4sources of SB623 cells from which conditioned medium was obtained; “SD”refers to standard deviation. “—” indicates that levels, if any, werebelow the limit of detection in the assay; “n/a” indicates “notapplicable”

Example 8 Effect of an Inhibitor of VEGF Signaling on HUVEC Viabilityand Proliferation

In light of the large amounts of VEGF secreted by both MSCs and SB623cells, the contribution of VEGF to the pro-angiogenic activities of MSC-and SB623-conditioned media was tested using an inhibitor of VEGFsignaling. SU5416 (VEGFR2 kinase inhibitor III, EMD Millipore,Billerica, Mass.) blocks downstream signaling by VEGF receptor 2 (Flk-1)and, to a lesser extent, by VEGF receptor 1 (Flt-1) and other receptortyrosine kinases, thereby inhibiting angiogenesis.

HUVEC viability assays (propidium iodide uptake and Bcl-2 expression)were conducted as described in Example 2 on two batches of SB623cell-conditioned medium, in the presence and absence of 50 nM SU5416except that cells were cultured for live days, instead of seven, beforeassay. The inhibitor was added to cultures 30 minutes before addition ofCM. Since higher concentrations of SU5416 can inhibit receptor tyrosinekinases other than VEGFR2, this SU5416 concentration was chosen so thatVEGFR2 signaling (but not signaling by, e.g., PDGF receptor, EGFreceptors, or Flt3) was inhibited. The results, shown in FIGS. 10A and10B, indicate that more cells take up PI (FIG. 10A) and fewer cellsexpress the anti-apoptotic Bcl-2 protein (FIG. 10B) when HUVECs arecultured in SB623 conditioned medium and SU5416, than when they arecultured in SB623 cell-conditioned medium alone. Thus, inhibition ofVEGF receptor activity partially reduces the positive effect of SB623cell-conditioned medium on viability of HUVECs, pointing to a role ofthe VEGF protein in these effects.

The effect of the VEGF receptor inhibitor on stimulation of HUVECproliferation by SB623 cell factors was also assessed. Assays forexpression of Ki67 were conducted as described in Example 3, except that50 nM SU5416 was added to cultures 30 minutes before addition ofconditioned medium, and cells were cultured for five days, instead ofseven, before assay. The results, shown in FIG. 11 and averaged from twodonors, indicate that the enhancement of HUVEC proliferation observed inthe presence of conditioned medium from SB623 cells was partiallyreversed by inhibition of VEGFR2.

These results point to a role for VEGF, in addition to other SB623cell-derived factors, in the pro-survival and pro-proliferative activityof MSC and SB623 cell conditioned media.

Example 9 Effect of an Inhibitor of VEGF Signaling on Tube Formation byHuman Umbilical Vein Endothelial Cells (HUVECs)

HUVEC tube formation assays with conditioned medium from MSCs and SB623cells were conducted as described in Example 4, in the presence andabsence of the VFGF2 receptor inhibitor SU5416. Cells cultured in theabsence of conditioned medium were used as negative controls; and cellscultured in the presence of VEGF (10 ng/ml were used as positivecontrols. The results, shown in FIG. 12, indicate that VEGF,MSC-conditioned medium and SB623 cell-conditioned medium all promotetube formation; while the VEGFR2 inhibitor SU5416 reduces thestimulation of tube formation by all of these agents.

Quantitation of tube formation was conducted, as described in Example 4,for HUVECs exposed to SB623 cell conditioned medium in the presence andabsence of SU5416, at 16 and 40 hours after plating. The results, shownin FIG. 13, indicate that, at both time points, inhibition of VEGFR2completely reversed the positive effect of CM on tube formation.

Example 10 Effect of an Inhibitor of VEGF Signaling on Vessel Outgrowthand Branching in an Aortic Ring Assay

Aortic ring angiogenesis assays were conducted as described in Example 5on one batch of SB623 cell-conditioned medium, in the presence andabsence of 50 nM SU5416. The inhibitor was added to cultures 30 minutesbefore addition of CM and rings were assayed after 10 days of culture.The results indicate that the vessel outgrowth and branching resultingfrom culture of aortic rings in SB623 cell-conditioned medium (FIG. 14,compare left and center panels) was reduced in the presence of the VEGFreceptor inhibitor SU5416 (FIG. 14, compare center and right panels).These results provide fu Cher evidence for the role of VEGF in thepro-angiogenic activities of SB623 cell-conditioned medium.

The results obtained using a VEGF receptor inhibitor (described above),while confirming the importance of VEGF to these processes (particularlytube formation, vessel outgrowth and vessel branching) do not rule outthe participation of additional factors (other than VEGF) in thepro-angiogenic activities of MSC- and SB623 cell-conditioned media.

What is claimed is:
 1. A method for treating an ischemic disorder in asubject, wherein the ischemic disorder occurs outside the centralnervous system, the method comprising: administering, to a site ofischemic disorder in the subject that is outside the central nervoussystem, a therapeutically effective amount of cells, wherein the cellsare made by a method comprising: (a) providing a culture of mesenchymalstem cells, (b) contacting the cell culture of step (a) with apolynucleotide comprising sequences encoding a Notch intracellulardomain (NICD) wherein said polynucleotide does not encode a full-lengthNotch protein, (c) selecting cells that comprise the polynucleotide ofstep (b), and (d) further culturing the selected cells of step (c) inthe absence of selection and in the absence of trophic factors; whereinthe cells produce angiogenin, angiopoietin-2 (ANG-2) and heparin-bindingepidermal growth factor-like growth factor (HB-EGF) at higher levelsthan do the mesenchymal stem cells of step (a).
 2. The method of claim1, wherein the ischemic disorder is selected from the group consistingof cardiac ischemia, ischemia of the bowel, ischemia of the limb,cutaneous ischemia, retinal ischemia and cerebral palsy.
 3. The methodof claim 2, wherein the ischemia of the bowel is ischemic colitis ormesenteric ischemia.
 4. The method of claim 1, wherein the cells produceone or more additional angiogenic factors selected from the groupconsisting of basic fibroblast growth factor, leptin, platelet-derivedgrowth factor-BB, hepatocyte growth factor, vascular endothelial growthfactor and placental growth factor.
 5. The method of claim 1, whereinadministration results in augmentation of blood vessel sprouting.
 6. Themethod of claim 1, wherein administration results in prevention ofendothelial cell death.
 7. The method of claim 1, wherein administrationresults in enhancement of endothelial cell survival.
 8. The method ofclaim 1, wherein administration results in stimulation of endothelialcell proliferation.
 9. The method of claim 1, wherein administrationresults in enhancement of vessel branching.
 10. A combination for use intreating an ischemic disorder in a subject, wherein the ischemicdisorder occurs outside the central nervous system, wherein thecombination comprises: (a) a first pro-angiogenic agent comprising cellsmade by a method comprising: (i) providing a culture of mesenchymal stemcells, (ii) contacting the cell culture of step (i) with apolynucleotide comprising sequences encoding a Notch intracellulardomain (NICD) wherein said polynucleotide does not encode a full-lengthNotch protein, (iii) selecting cells that comprise the polynucleotide ofstep (ii), and (iv) further culturing the selected cells of step (iii)in the absence of selection and in the absence of trophic factors;wherein the cells produce angiogenin, angiopoietin-2 (ANG-2) andheparin-binding epidermal growth factor-like growth factor (HB-EGF) athigher levels than do the mesenchymal stem cells of step (i); and (b) asecond pro-angiogenic agent.
 11. The combination of claim 10, whereinthe second pro-angiogenic agent is a polypeptide.
 12. The combination ofclaim 11, wherein the polypeptide is a transcription factor thatactivates transcription of a pro-angiogenic protein.
 13. The combinationof claim 12, wherein the pro-angiogenic protein is vascular endothelialgrowth factor (VEGF).
 14. The combination of claim 13, wherein thetranscription factor is a non-naturally-occurring zinc finger proteinthat activates transcription of the VEGF gene.
 15. The combination ofclaim 10, wherein the second pro-angiogenic agent is a nucleic acid. 16.The combination of claim 15, wherein the nucleic acid encodes atranscription factor that activates transcription of a pro-angiogenicprotein.
 17. The combination of claim 16, wherein the pro-angiogenicprotein is vascular endothelial growth factor (VEGF).
 18. Thecombination of claim 17, wherein the transcription factor is anon-naturally-occurring zinc finger protein that activates transcriptionof the VEGF gene.
 19. A method for use in treating an ischemic disorderin a subject, wherein the ischemic disorder occurs outside the centralnervous system, the method comprising administering to the subject atherapeutically effective amount of the combination of claim
 10. 20. Themethod of claim 19, wherein administration results in augmentation ofangiogenesis.
 21. The method of claim 20, wherein said augmentation ofangiogenesis comprises a process selected from the group consisting ofone or more of prevention of endothelial cell death, enhancement ofendothelial cell survival, stimulation of endothelial cellproliferation, enhancement of blood vessel sprouting and enhancement ofblood vessel branching.